Fatty acids, coco, tetraesters with bis[2,2-bis(hydroxymethyl)butyl] adipate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between February 3, 2011 and February 28, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,6-bis({2,2-bis[(undecyloxy)methyl]butyl}) hexanedioate

EC Number:

700-772-5

Cas Number:

1190961-28-4

Molecular formula:

N/A - too complex

IUPAC Name:

1,6-bis({2,2-bis[(undecyloxy)methyl]butyl}) hexanedioate

Test material form:

other: liquid

Details on test material:

- Physical state: Clear straw liquid
- Analytical purity: > 99%
- Lot/batch No.: 1460-009-08-135-2
- Storage condition of test material: room temperature

Method

Target gene:

Not required

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Non-mammalian study

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Non-mammalian study

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital (PB, 4 times 0.03-0.06g/kg/day) and 5,6-benzoflavone (BF, I time 0.08g/kg/day) induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1: Range-finding test
Salmonella strains: 4.88, 19.5, 78.1, 313, 1250, and 5000 µg/plate


Experiment 2: Main test
All Salmonella strains (with and without S9) except TA100 (without 156: 313, 625. 1250 150, 500, 1500, 5000 μg/plate.
Salmonella 00 (without S9): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
E.coli strain WP2uvrA- : 50, 150, 500, 1500, 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, suspended in dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in-house. Acetone was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

no

Positive controls:

yes

Remarks:

Concurrent

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

no

Positive controls:

yes

Remarks:

Concurrent

Positive control substance:

9-aminoacridine

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

no

Positive controls:

yes

Remarks:

Concurrent

Positive control substance:

sodium azide

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre-incubation method at multiple dose levels, in duplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors).
The assay was performed by mixing 0.1 ml of bacterial culture, 0.05 ml of the substance formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar. In total six concentrations of the test material and a vehicle control (acetone) were tested. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
DURATION
- Preincubation period: 20 min
- Exposure duration: Approximately 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A


SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: 2 replicates of each strain at each concentration both in the presence and absence of S9

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible and dose-related increase in the revertant count in at least one strain of bacteria.

Statistics:

NDA

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Cytotoxicity to bacteria by the test material was not observed for TA98, TA100, TA1535, TA1537 and WP2uvrA at any dose level with or without metabolic activation in the dose-determination test and the mutagenicity test.

Precipitate of the test material was observed at 5000ug/plate for TA98, TA100, TA1535, TA1537 and WP2uvrA with metabolic activation in the dose-determination test and the mutagenicity test.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains at any dose level, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The substance was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Method

This study was designed to assess the mutagenic potential of the test substance using a bacterial/ microsome test system.

Salmonella typhimurium strains TA98, TA100, TA1535, TAI537  and Escherichia coli WP2uvrA were treated with the test material using the pre-incubation method at six dose levels, in duplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for the dose-determination test was 4.88 to 5000 ug/plate. The experiment as repeated on a separate day using the dose range, 156 to 5000 ug/plate, fresh cultures of the bacterial strains and fresh test material formulations.

Results

Cytotoxicity to bacteria by the test material was not observed for TA98, TA100, TA1535, TA1537 and WP2uvrA at any dose level with or without metabolic activation.

Precipitate of the test material was observed at 5000 ug/plate for TA98, TA100, TA1535, TA1537 and WP2uvrA with metabolic activation.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

Conclusion

The test material was considered to be non-mutagenic under the conditions of this test.